The optical scanning microscope is a combination of optical microscopy, with its long history, and electronics, which has made rapid progress in recent years, and its usefulness is widely recognized, especially with the development of the confocal point optical system. One of the advantages of the confocal optical system is that it can provide a marked improvement in image resolution and contrast by eliminating unwanted scattered light.
The basic principle of the optical scanning microscope and confocal optical system was described over thirty years ago (see Reference (1): U.S. Pat. No. 3,013,467). In the systems of the time, an ordinary lamp was used as the light source and scanning was effected by using a voice coil mechanism to move the specimen stage, which made it difficult to quickly produce clear images.
With the aim of providing real-time image capability, a confocal optical microscope that used a Nipkow disk perforated with a large number of holes was developed a considerable time ago (see for example Reference (2): Journal of the Optical Society of America Vol. 58 (1968) pp 661 to 664; U.S. Pat. No. 3,517,980; and U.S. Pat. No. 4,802,748). This system, which was referred to as tandem scanning, could provide real-time naked-eye or photographic observation of images without using optoelectronic conversion, and received some practical application. However, a critical drawback of the tandem and other Nipkow disk system confocal microscopes has been that the light source was not bright enough, and a further problem was that with some observation objects the confocal effect was too small. These drawbacks have continued to limit the application of such systems.
With the emergence of lasers, with their high spatial coherence, lasers have also come into use as light sources for confocal microscopes. In the first laser scanning microscopes (LSMs) the movement of the laser beam was effected by moving the objective lens in close proximity to the specimen (see Reference (3): Nature Vol. 223 (1969) p831; Applied Optics, Vol. 10 (1971) pp 1615 to 1619). Since then the development of laser scanning microscopes has resulted in many improvements that have broadened the range of application of such instruments to a number of fields including mineralogy, semiconductors, medicine, biology and cytology.
LSMs are divided broadly into types in which the specimen stage is scanned and types in which the laser beam is scanned. Some systems use a polygonal mirror rotating at high speed for linear laser beam scanning for cytological examinations (see Reference (4): The Journal of Histochemistry and Cytochemistry Vol. 27 (1979) pp 153 to 159; Analytical and Quantitative Cytology Vol. 3 (1981) pp 55 to 66). However, in recent years most commercial LSMs are stage scanning systems or beam scanning systems that incorporate a mirror galvanometer and acousto-optical deflector (AOD) or the like (see Reference (5): Scanning Vol. 7 (1985) pp 79 to 87 and pp 88 to 96). Compared to beam scanning systems, stage scanning systems are advantageous in terms of objective lens distortion and simplicity of the apparatus; in some cases the focus of development has been aimed at modifying the stage itself (see Reference (6): JP-A-62-17723). Two-dimensional laser beam scanning by mirror galvanometer is used by many LSMs, but there have been many announcements of systems using AODs as a way of attaining higher scanning speeds.
For example, a non-confocal system transmission type LSM has been announced which uses two AODs arranged at right angles to scan a laser beam two-dimensionally (see Reference (7): JP-A-61-248023; Applied Optics Vol. 25 (1986) pp 4115 to 4121). A slit confocal type LSM has been announced which uses a total of three scanning systems, an AOD and a mirror galvanometer used to scan the beam in two dimensions and a CCD line sensor as the light receiving means (see Reference (8): JP-A-61-80215; SPIE Proceedings Vol. 765 (1987) pp 53 to 60). A pinhole confocal system has also been realized in a reflecting microscope in which an AOD and mirror galvanometer are used to provide two-dimensional scanning (see Reference (9): JP-A-63-298211; SPIE Proceedings Vol. 809 (1987) pp 85 to 88). A reflecting and transmitting type confocal system has also been developed using two AODs at right angles and a non-storage type image dissector tube (IDT) (see Reference (10): U.S. Pat. No. 4,827,125; SPIE Proceedings Vol. 1161 (1989) pp 268 to 278).
However, a major drawback with such LSMs is the difficulty of the laser beam scanning control method. The stage scanning LSM of Reference (6), for example, performs excellently when the specimen of interest is something stationary, such as a mineral sample, but is entirely unsuitable for viewing a live biological specimen moving in a solution. For observation of live biological specimens, it is necessary to use a high-speed laser beam scanning that is compatible with a standard TV scanning rate.
The system described in Reference (4) which uses a polygonal mirror deflector is advantageous in terms of high scanning speed and linearity, and can be made compatible with normal video rates. However, the high-speed polygonal mirror, which uses pneumatic bearings, makes the system apparatus large and costly, and there are other problems such as the service life and reliability of the bearings, shaft run-out and facet trueness and, in a high magnification power microscope, the difficulty of excluding the effects of the vibration produced by the rotation of the deflector itself, all of which combine to make such a system less than fully practicable.
In the mirror galvanometer LSM of Reference (5), the use of a resonance-vibration mirror provides some degree of high-speed scanning capability. With a mirror frequency set at 8 kHz, for example, it should be possible to follow a standard TV horizontal scan frequency (about 16 kHz). However, the rapid wear on mirror suspension bearings caused by such a high resonant frequency has an adverse affect on system durability. Over time, shaft wear and fatigue can result in shaft run-out, deviation, hysteresis and other such variation, which, in the case of an LSM in which image quality depends on the beam scanning precision, degrades the reliability of the system itself.
A system that uses the precision scanning performance of the non-oscillation AOD for high-speed scanning can be readily adapted to a standard television horizontal scanning frequency of 15.75 kHz. As the vertical scanning frequency of 60 Hz is readily attainable even with a mirror galvanometer or scanning stage, the incorporation of an AOD permits images of living biological specimens to be viewed at video picture frequency in real-time on a TV monitor. However, a drawback of acousto-optical deflectors is that as they are diffraction devices, and the wavelength dependency of the diffraction can produce color dispersion, and also, owing to polarization plane constraints, it is difficult to apply AODs to confocal fluorescent microscopes which use the acquisition of weak fluorescent light of different wavelengths produced when cells or the like are excited by the laser beam.
A system described in Reference (9) attempts to tackle this using an AOD and pinhole to detect the fluorescent component. In the system configuration used for this, piezoelectric devices are used to control the three-dimensional spatial positioning of the pinhole, but owing to various constraints such as the AOD transmission loss and the Bragg angle dependency of the incident light, optical system settings are not easy.
By using a CCD line sensor or IDT as the detector as in References (8) and (10), the detected fluorescent light is not passed through the AOD, thereby avoiding such problems. However, the fact that the CCD line sensor is a stored charge device means that in the case of a confocal system of a microscope, resolution cannot be improved in the CCD scanning direction. Additionally, since the detector is incapable of providing higher sensitivity, such a system is not suited to applications involving the detection of weak fluorescent light. Moreover, while the use of an IDT makes it possible to configure a confocal system that also offers high sensitivity during detection of fluorescent light, the fact that the laser beam scanning and synchronization systems are complex and that IDTs are very costly makes the practicality of such systems questionable.
The biggest drawback of an AOD deflector is that when high image resolution is a specific requirement, the AOD has to be constituted of a special substance such as TeO.sub.2 or PbMoO.sub.4, formed into a flat, uniform optical medium with a large-diameter aperture. This usually requires the disposition of an anamorphic lens, which is complex to adjust, at the front and rear of the AOD, and the crystal itself is far more costly than small-aperture media.
With reference to the cost aspect, a high-speed vibration mirror is costly, and a high-precision, high-speed polygonal mirror with pneumatic bearings is very costly. This, together with problems concerning deflector reliability and ease of use, is probably what has hindered the practical use and spread of LSMs which permit real-time observation.
Recent years have also seen the realization of high-definition television (HDTV), the aim of which is to provide improved resolution and picture quality, and the feasibility of HDTV compatible LSM systems is being studied. However, with an HDTV system having a horizontal scanning frequency of 30 kHz or more, the above-described problems of each type of deflector arrangement would be correspondingly magnified if attempts were made to operate them at such a high frequency. For this reason, despite the interest in the potential of a HDTV compatible LSM, specific working principles or methods for a commercially practicable system have not yet emerged.
The object of this invention is to provide a laser scanning microscope in which the deflector control arrangement is highly reliable and offers long service life and high scanning precision and stability even under high scanning frequency operation, and in which the confocal system can be arranged as a reflection or transmission mode system and can readily be used to detect weak fluorescent light having a different wavelength from that of the scanning laser beam, and in which the optical deflector can be configured at low cost, and can be adapted to high-definition television.